Laboratory Diagnosis of Bacterial Infections

Laboratory Diagnosis of Bacterial Infections

Brenda C. Love and Robert L. Jones

The purpose of clinical bacteriology is to provide rapid and accurate information concerning the presence or absence of a bacterial agent in an infectious disease process. It usually requires at least 24 to 72 hours for bacteria to be isolated, but the clinician can seldom wait that long to institute therapy. Knowledge of the prevalence of specific pathogens responsible for defined clinical syndromes and trends in antimicrobial susceptibility patterns provides the basis for making rational early treatment decisions and selecting the antimicrobial agent most likely to be effective. This knowledge can only be developed and updated by submission of specimens to the laboratory for isolation, identification, and antimicrobial susceptibility testing. Therefore, the use of the laboratory builds a database that can guide the design of treatment plans for future as well as current patients.

Use of the microbiology laboratory is subject to unique pitfalls posed by the diversity of bacterial agents, each with unique requirements for identification: multiple and often poorly accessible sites for collection of specimens, contamination of specimens with indigenous flora, and the necessity for subjective interpretation of results. Problems in communication may occur between the microbiology laboratory and the clinician regarding the suitability of the specimen for laboratory examination, uncertainty of the significance of bacterial isolates, interpretation of reports, and length of delay between the submission of the specimen and the production of needed information. Special efforts by the clinician to provide history and signalment information along with the specimen assist the laboratory in recognizing and reporting significant results.

Major technologic advances have changed the way microbiology laboratories function. Bacterial identification is now achieved largely through the use of miniaturized packaged systems, many of which are automated. Antimicrobial susceptibility testing is commonly accomplished with a commercial microdilution system or some form of instrumentation. Immunodiagnostic methods for antigen and antibody detection have benefited greatly from radioimmunoassay, direct and indirect fluorescent antibody (FA) testing, enzyme-linked immunosorbent assay, latex agglutination, and immunoblotting, as well as the development of monoclonal antibody technology. Nucleic acid technology is leading the clinical microbiology laboratory into a new era of molecular diagnostics for detecting and identifying microorganisms. Cost constraints for new technology may limit the scope of services that can reasonably be provided by any one laboratory. Therefore, it is unreasonable to expect equal diagnostic capabilities from all microbiology laboratories for all microorganisms. With increasing frequency, specimens must be sent to reference laboratories that perform specialized diagnostic tests. Web Appendix 5 lists commercial, state, and federal diagnostic laboratories that provide specific microbiologic testing for infectious diseases of dogs and cats.

Diagnostic Methods

Microbiologic tests complement clinical judgment, enhance the clinician’s ability to select specific antimicrobial drugs, and ultimately improve patient care through detection and identification of the etiologic agents.

Direct Microscopic Examination

Direct microscopic examination of exudates or infected body fluids is the single most important and cost-effective laboratory procedure that can be done for diagnosis and management of bacterial infections. Evaluations can be done in various clinical settings to obtain immediate information on the number, morphologic characteristics, and Gram-staining properties of microorganisms (Table 29-1) and the host cellular response. Purulent inflammatory responses are most suggestive of bacterial infection. Microscopic examination also gives an indication of the suitability of the specimen for culture, the likelihood of the presence of infection, the likely pathogens, and the predominant organisms in a mixed infection. This information may be used as a basis for interpretation of the significance of subsequent culture results.

Bacteria are readily observed in a smear from a specimen when they occur in a concentration of about 104 to 105 per milliliter. Examination of specimens such as blood or cerebrospinal fluid (CSF) for bacteria is usually unrewarding because even when present, the bacteria are too few in number to be detected. Some bacteria, such as spirochetes and mycoplasmas, do not stain well with Gram stain. Dark-field microscopy allows visualization of spirochetes, but mycoplasmas are too small for observation by light microscopy. Background material and artifacts can interfere with interpretation. Mucus or other proteinaceous matter that stains gram negative may cause slender, weakly staining bacilli to blend into the background, making them difficult to visualize. Differential staining techniques that enhance visualization of gram-negative bacteria include methylene blue, Giemsa, and Wright stains.

Isolation and Identification

Whether isolation and identification are attempted depend on many factors, the most important of which are the source of the specimen and whether or not a presumptive diagnosis can be made microscopically. Most commonly encountered pyogenic bacteria readily grow on routinely inoculated blood agar plates. Liquid media provide enrichment for recovery of organisms present in small numbers and facilitate isolation of fastidious organisms. In some cases, selective media must be used to suppress growth of contaminants and normal flora while allowing growth of the pathogen.

Certain infectious diseases may be more efficiently diagnosed by direct detection of antigens, nucleic acids, or toxins. For diagnosis of some clostridial diseases, such as botulism (see Chapter 40), tetanus (see Chapter 41), and enterocolitis (see Chapter 37), it is imperative to identify the toxin rather than rely solely on isolation of the bacteria. Direct or indirect FA stains are extremely helpful for the rapid presumptive diagnosis of infection with some bacteria. Because Yersinia pestis (see Chapter 45) and Francisella tularensis (see Chapter 46) are hazardous to laboratory personnel, antigen detection by direct FA staining of exudate or tissue is the best tool for their rapid and specific identification. DNA probes and nucleic acid amplification techniques are useful for identification of microorganisms for which culture and serologic methods are difficult, extremely expensive, or unavailable.

Specimen Collection

Specimens must be collected from the actual site of infection with a minimum of contamination from adjacent tissues, organs, or secretions (Fig. 29-1). They must then be transported to the laboratory without further contamination or change in the relative numbers of bacteria. Some specimens are likely to become contaminated with indigenous flora during the collection process. Great care must be taken to use aseptic technique when collecting specimens to reduce the amount and likelihood of contamination. Some specimens are simply inaccessible except by aspiration or biopsy of deeper tissues. For all specimens obtained by biopsy or fine-needle aspiration, skin decontamination should be performed as it is before surgery. Specimens should be collected as early in the course of the disease process as possible. As the disease progresses and necrosis of tissue occurs, some microorganisms may die or be overgrown by other bacteria.

Whenever possible, specimens should be obtained before the administration of antimicrobials. However, their use does not necessarily preclude the recovery of bacteria from selected tissues in which low antimicrobial concentrations are achieved or from those animals infected with antimicrobial-resistant bacteria. If it is not possible to collect the specimen before any antimicrobials are administered, the specimen should be collected just before the next dose is given. If antimicrobials are concentrated at the sampling site, such as during urine collection, it is best to wait 48 hours after the last dose before collecting a specimen.

An adequate quantity of material (several millimeters or grams) should be obtained for appropriate laboratory tests. All too frequently, an inadequate amount of material is obtained with a swab, making it nearly impossible for the laboratory to make appropriate smears and inoculate adequate culture media. A swab should never be submitted in lieu of curettings, biopsy material, fluid (especially urine), or surgically removed tissue.

Multiple specimens should be submitted when lesions are present at several sites or when more than one laboratory procedure is requested. Multiple blood samples are necessary for detection of bacteremia. Multiple fecal samples are also indicated for detection of enteric pathogens such as salmonellae.

Collection Devices and Transport

Appropriate collection devices and specimen transport systems are needed to ensure survival of the microorganism without overgrowth and for optimal isolation and identification. Various containers are commercially available, ranging from simple swab and plastic tube combinations to complicated specimen collection devices (Fig. 29-2). Swabs must be made of noninhibitory materials and transported in a sterile container. Many bacteria are susceptible to desiccation during transport. Swabs are only acceptable for transport of specimens when provided with a humidified transporting chamber or placed in transport medium. Several swab-transport medium systems exist and are often available from the clinical laboratory. Tissue, exudate, feces, or fluid should be submitted in an appropriate closed (leak-proof), sterile container. Plastic screw-cap cups and blood collection tubes that do not contain anticoagulants or preservatives are examples of recommended specimen containers. Each tissue or specimen must be placed in a separate container. Direct aspiration into a syringe is often a convenient and satisfactory means for of tissue and fluid collection. However, the needle must be removed for transport to avoid injuries, and the syringe should be capped.

Various types of media may be used for transporting specimens to the laboratory. Transport media such as Stuart’s, Cary-Blair, Amies, and anaerobic devices (frequently available from the microbiology laboratory) are buffered nonnutritive formulations that preserve the viability of fastidious organisms in the specimen as well as minimize overgrowth by rapidly growing bacteria that may be present. The ordinary nutrient type of broth such as that in blood culture bottles may be used only when swabs or aspirates are collected from normally sterile body sites (e.g., CSF, synovia) and when great care has been taken to avoid any contamination. Special anaerobic transport devices must be used to prevent exposure of obligate anaerobes to lethal concentrations of oxygen (see Chapter 39). Because reduced oxygen is not lethal for the aerobes and facultative anaerobes, they can be transported in the same anaerobic transport devices. Tissue in formalin, dry swabs, and urine collected several hours earlier and not refrigerated are examples of specimens that are unsuitable for bacterial culture.


Successful isolation of microorganisms from blood requires an understanding of the intermittence and low order of magnitude of most bacteremias (see Blood Culture, Chapter 86, for the appropriate technique). Several specimens must be collected for culture over a period of time. If possible, the first blood culture specimen should be drawn at the onset of fever. Another suggestion is to take three or four cultures within 1.5 to 3 hours; if more than one culture yields the same organism, it is probably significant.

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Aug 6, 2016 | Posted by in INTERNAL MEDICINE | Comments Off on Laboratory Diagnosis of Bacterial Infections

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